Yukiya Maruguchi

A new skin equivalent : keratinocytes proliferated and differentiated on collagen sponge containing fibroblasts

Three types of artificial skin containing keratinocytic components were prepared and tested for comparison. Keratinocytes were cultured on the artificial skin dermis (collagen sponge) by the air-liquid interface culture method. In order to create continuous keratinocytic layers on the artificial skin dermis, pores of its uppermost layer were filled beforehand with type I collagen gel, Matrigel, or fibroblasts. A band of keratinocytes consisting of two to six cell layers was formed on collagen gel-coated artificial skin dermis. On Matrigel-coated artificial skin dermis, keratinocytes were piled up into about 20 cell layers, but cell differentiation was incomplete; cornified material was not fully developed, and the proportion of cuboidal cells was very high compared with normal epidermis. Keratinocytes formed continuous layers on the fibroblasts-artificial skin dermis complex without gel coating. Keratinocytes proliferated well and differentiated properly on this matrix, and their histologic appearance was similar to that of normal epidermis. Thus keratinocytes cultured on the fibroblast-artificial skin dermis complex seem to be a good skin equivalent.

Review of acellular and cellular artificial skins.

Following the development of a bilayer acellular artificial skin by Yannas et al. which seemed unique with respect to spontaneous conversion of the inner collagen sponge into a dermis-like connective tissue layer, we started to develop an alternative acellular artificial skin and extended the indications for the material. Since reporting our early results of experimental and clinical use of the original version of the acellular artificial skin, several improvements have been made in stages to eliminate some drawbacks related to disinfection and preservation and to reduce the primary cost of manufacture. We used this material on 51 skin defects in 39 patients with success. The latest version of the material was also evaluated in multicenter clinical trials involving 80 cases. Separately, a material capable of sustained release of an antibiotic was developed and used in 6 wounds prone to infection with success. To solve the problem of two-stage surgery, a cellular artificial skin composed of an outer keratinocytic layer and an inner collagen sponge containing fibroblasts was produced using cell culture method by modifying the technique proposed by Boyce et al. This report reviews our acellular and cellular artificial skins.

Establishment and characterization of a novel in vitro angiogenesis model using a microvascular endothelial cell line, F-2C, cultured in chemically defined medium

SummaryThe behavior of vascular endothelial cells (EC) is an important factor in the processes involved in angiogenesis, but the regulatory mechanisms of angiogenesis, especially underlying the tubulogenesis by EC are not yet clear. Although a number of in vitro experimental models of tubulogenesis have been developed by use of cultured EC, most of those models are too complex to be easily handled and further, the culture media are usually supplemented with serum, creating problems in interpretation of experimental results. To generate a simple in vitro angiogenesis study model under serum-free culture conditions, we adapted a murine microvascular endothelial cell line, F-2, to a chemically defined medium, Cos Medium 001, and successfully established a subline of F-2, designated F-2C, which revealed a unique growth pattern. In Cos Medium 001, F-2C proliferates in a cobblestone pattern at an early growth stage, but, at a late growth stage, spontaneously differentiates to form three-dimensional honeycomblike tubular structures without the supplementation of any specific factors. The cell aggregation activity of F-2C in the presence of Ca2+ was much greater than that of F-2. The amount of subendothelial matrix deposited by F-2C was significantly higher than that by F-2, and increased prominently after the F-2C cells reached the differentiating stage of tubulogenesis. These findings indicate that F-2C is a new EC line in which tubulogenesis is spontaneously induced by the marked deposition of basement membrane analog to the subendothelial matrix and by the enhancement of presumable cadherin activity. We suggest that this cell line, F-2C, represents a simple and useful in vitro angiogenesis model.

Survival period of tumor-bearing mice is prolonged after the interferon-γ-producing gene transfer

A highly tumorigenic keratinocyte-derived carcinoma cell line, designated as Pam-T, was established from a Pam212 line. The intradermal injection of more than 10(5) of these cells into syngeneic BALB/c mice induced substantial tumors. The tumors progressively enlarged and then invaded the peritoneal cavity leading to the death of the host mice. To comprehensively investigate the effects of interferon-gamma on tumorigenicity, we manufactured interferon-gamma-producing PamT cells by interferon-gamma gene transfer and examined the characteristics of the tumors induced by these cells in syngeneic mice. Interferon-gamma producing cells exhibited an apparently similar in vitro cell growth pattern and in vivo tumor formation to control cells, but the mean survival of the mice with the interferon-gamma-producing cells was significantly longer compared with control mice.

Morphological Characterization of Hemangiomatous Tumors Derived from a Novel Murine Vascular Endothelial Cell Line (F-2)

Hemangiomatous tumors were induced in Balb/c nude mice by inoculating F‐2 cells (5 times 106) from a novel tumorigenic murine endothelial cell line which had been established and maintained in our laboratories. These tumors were morphologically investigated in the course of development. Subcutaneous hemorrhage was observed at the inoculation site within 12 hours after the injection of F‐2 cells, followed by development of skin tumors of various sizes at the same sites. They were dome‐shaped, glossy surfaced, black, soft tumors. Mice finally died of massive blood loss due to internal and/or external hemorrhage (on day 10–77). Light microscopically, F‐2 cells formed aggregates immediately after inoculation and then branched into a network of channels and cysts containing erythrocytes. Thereafter, spongiform structures composed of various sizes of cysts appeared with subsequent formation of a single large blood‐filled cyst lined by one or two layers of thin cells. Under an electron microscope, F‐2 cells, possessing large amounts of cytoplasm, formed narrow spaces which were occasionally incomplete with ambiguous basal lamina at the early stages. However, later, in large cysts, they became attenuated, tightly connected, and produced complete lumen surrounded by a basal lamina. Immuno‐histological demonstration of H‐2 K, D antigen showed that tumors induced by F‐2 cells mainly consisted of the inoculated F‐2 cells. These results indicate that F‐2 presents a good experimental system for investigation of vascular endothelial cell tumorigenesis and differentiation.

Treatment of inflammatory facial acne vulgaris: Comparison of the 1450‐nm diode laser and conventional physical treatment

The efficacy of the 1450‐nm diode laser in the treatment of inflammatory facial acne was evaluated by comparing it with conventional physical treatment. Seventeen patients received laser treatment on the right side of the face and conventional physical treatment on the other side. The two modalities were compared through photographs, inflammatory acne lesion counts, and a patient questionnaire. Clinical response was evaluated in 16 patients. Evaluation of baseline and follow‐up photographs indicated that more improvement was obtained after laser treatment than by physical treatment in six patients. In two patients, physical treatment yielded better results than laser treatment. Equal effect was obtained in eight patients. All patients had a reduction in the inflammatory acne lesion count on the laser‐treated side, which was statistically significantly greater on the laser‐treated side compared with the side treated physically (p = 0.039, Wilcoxon signed ranks test). By the assessment of patient satisfaction, seven patients preferred laser treatment, two patients preferred physical treatment and three patients found laser treatment equal to physical treatment. Questionnaire details could not be obtained in 4 patients. This study indicates that the 1450‐nm diode laser is a new option for local treatment of acne.

Modulatory effects of interferon-γ on the fibronectin receptor function of squamous cell carcinoma cells in vitro

We previously showed that the in vivo invasion of a squamous cell carcinoma induced by the intradermal injection of tumor cells was significantly delayed after the IFN-gamma-producing gene transfer to tumor cells. With respect to the mechanism of the delayed invasion, it was suggested that the IFN-gamma might inhibit the adhesion of the cells to extracellular matrices (ECM) and the subsequent locomotion. Thus, we examined the effect of IFN-gamma on the adhesion of Pam-T cells to ECM. The attachment of Pam-T cells to fibronectin (FN) was significantly higher than that to laminin (LN), collagen type I (COL I) or collagen type IV (COL IV) substrata. The attachment to FN was significantly enhanced specifically by the IFN-gamma-treatment of the cells, although the attachment to LN, COL I or COL IV was not altered by IFN-gamma. Neither IFN-alpha nor IFN-beta had any effect on the attachment of Pam-T cells to FN. When Pam-T cells were treated with IFN-gamma together with a neutralizable anti-IFN-gamma antibody, this enhancement was completely abolished. Moreover, the attachment of IFN-gamma-treated Pam-T cells as well as non-treated cells to FN was blocked by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), but not by the control peptide Arg-Gly-Glu-Ser. Based on these results, we conclude that IFN-gamma specifically enhances the adhesiveness of Pam-T cells to FN substrata by the modulation of integrin activity.